Members of this superfamily are commonly referred to as “inverzincins,” because they feature a zinc-binding motif that is inverted with respect to that within conventional zinc-metalloproteases. Like insulin, IDE is structurally distinctive, consisting of two bowl-shaped halves connected by a flexible linker that can switch between “open” and “closed” states. In its closed state, IDE completely encapsulates its substrates within an unusually large internal cavity that appears remarkably well-adapted to accommodate insulin. IDE degrades several other intermediate-sized peptides, including atrial natriuric peptide, glucagon, and the amyloid b-protein; however, unlike insulin, most other IDE substrates are known to be hydrolyzed by multiple proteases. Diabetes melittus is a life-threatening and highly prevalent group of endocrinological disorders that, fundamentally, are characterized by impaired insulin signaling. Correspondingly, it is the common goal of most anti-diabetic therapies to enhance insulin signaling, either by direct injection of insulin, by stimulating the production or secretion of endogenous insulin, or by activating downstream targets of the insulin receptor signaling cascade. In principle, it should be possible to enhance insulin signaling by inhibiting IDE-mediated insulin catabolism. Pharmacological inhibitors of IDE in fact attracted considerable attention in the decades following the discovery of IDE in 1949. Quite significantly, a purified PR-171 inhibitor of IDE was found to potentiate the hypoglycemic action of insulin in vivo as early as 1955. Despite more than 60 years of research on IDE and its involvement in insulin catabolism, the development of smallmolecule inhibitors of IDE has proved to be a surprisingly elusive goal. We describe herein the design, synthesis, enzymologic characterization, and enzyme-bound crystal structure of the first potent and selective inhibitors of IDE. In addition, we show that inhibition of IDE can potentiate insulin signaling within cells, by reducing the catabolism of internalized insulin. These novel IDE inhibitors represent important new pharmacological tools for the 
experimental manipulation of IDE and, by extension, insulin signaling. Furthermore, our results lend new support to the old idea that pharmacological inhibition of IDE may represent an attractive approach to the treatment of diabetes mellitus. The development of Ii1 which potently inhibits IDE, but not cathepsin D enabled us for the first time to address cleanly this longstanding controversy. To that end, we conducted live-cell imaging of CHO-IR cells loaded with fluorescent insulin labeled exclusively at the Nterminus of the B chain with fluorescein isothiocyanate, a modification that has been shown not to interfere with binding to the IR. FITC-ins-loaded cells were washed then monitored for changes in PD 0332991 fluorescence in the presence of Ii1 or vehicle. In vehicle-treated cells, intracellular fluorescence decreased and extracellular fluorescence increased monotonically with time. By contrast, both intra- and extracellular fluorescence remained essentially constant in the presence of Ii1. Consistent with previous studies of insulin catabolism, the fluorescent species secreted by vehicle-treated cells were confirmed to be proteolytic fragments of FITC-ins. These results strongly suggest that the catabolism of internalized insulin is primarily, if not exclusively, carried out by IDE. Taken together, these results suggest correspondingly, insulin signaling can be potentiated significantly by inhibiting IDE proteolytic activity.